Combined cytogenetic and molecular genetic approaches will lead to the recognition of important genes and gene alterations that have significant clinical application in the control of cancer. Although integration of human papillomavirus (HPV) DNA in the cellular genome does not result in virus production, HPV integration is a hallmark of cervical carcinoma. HPV integration at chromosome fragile sites and near proto-oncogenes demonstrated by our studies suggests both a structural requirement and a biological selection of the phenotype. Several integration sites from HPV-immortalized keratinocyte lines were characterized by high-resolution in situ localization of viral DNA sequences relative to G-band and DNA replication patterns. HPV-16 sequences were mapped at G-light bands exhibiting late DNA replication. A system for digital imaging suitable for detection and analysis of a variety of immunofluorescent cytological reactions and for detection of single-copy genes or virus copies was introduced to investigate genomic alterations in carcinogenesis. By fluorescent in situ hybridization (FISH) and digital imaging, a single HPV-18 integration site was mapped in C4-1 cervical carcinoma. A DNase I- hypersensitive site was identified within 3 kb from the viral DNA. In addition, the integration region was undermethylated compared to tumor cell lines of other origins. Molecular and cytological evidence demonstrate that structural and functional chromatin features render specific genomic sites accessible to viral integration. FISH was optimized and used for detection of nuclear RNA on intact nuclei. In a Burkitt lymphoma cell line with a single-copy integrated Epstein-Barr virus, a specific hybridization signal reflecting the site of RNA transcription was directly visualized on intact nuclei. FISH in conjunction with enhanced digital processing allowed the detection and mapping of single-copy genes of relatively small size. A transforming gene (est) and a cholecystokinin-gastrin receptor gene were mapped in human chromosomes 10 (p11.2) and 11 (p15.5), respectively. Both genes may be involved in human carcinogenesis.